Synaptic transmission in brain is initiated by neurotransmitter release, which is mediated by synaptic vesicle exocytosis. Insight into the mechanisms of neurotransmitter release is essential for understanding how the brain processes information, and how synaptic transmission is affected in diseases such as Parkinson's disease, Alzheimer's disease, and drug addiction. Syntaxin is a SNARE protein on the presynaptic plasma membrane that is essential for neurotransmitter release. Syntaxin 1 functions at the membrane fusion step in exocytosis by interacting with a number of target proteins, but its precise mechanism of action remains incompletely understood. In the current application we propose a series of interdisciplinary experiments to examine the precise functions of syntaxin 1 in neurotransmitter release, and to relate this function to the role of other syntaxins in other membrane fusion processes. The proposed experiments unite structural and biophysical studies that combine multidimensional NMR techniques, biochemical approaches, and in vivo methods using PC12 cells and transgenic mice to achieve a comprehensive understanding of syntaxin 1 function. Five specific aims are proposed: (1) To determine the structure at atomic resolution of the "closed conformation" of syntaxin 1 and to study its conservation in other mammalian syntaxins. (2) To determine the structural basis for the interaction between syntaxin 1 and munc13-1, and to study the potential role of munc13-1 in promoting the conformational switch of syntaxin 1. (3) To determine the mode of binding of complexins to the core complex, and to study the consequences of this interaction on the properties of the core complex. (4) To study the in vivo function of syntaxin 1 in mice using transgenic and gene targeting methods. (5) To perform a systematic analysis of the structural properties of yeast syntaxins and of their interactions with proteins of the secl/muncl8 family. The results of this research will not only be important to understand the mechanisms of neurotransmitter release and membrane fusion in general, but will also have an impact in the design of therapies for brain disorders that involve changes in synaptic transmission.